Serum antibodies (Abs) with broad, anti-viral functions from HIV-infected patients generally map to more than one epitope. Many of these Abs target conserved regions of the HIV envelope, and, while elicitation of such Abs is desirable, inducing such Abs has proven difficult. An alternative, and perhaps more practical, approach is to induce Abs which, individually, display more limited breadth but which, together, display neutralizing and/or Ab-dependent cell-mediated anti-viral effects against a broad spectrum of viruses. Recent data suggest that Abs specific for the sequence-variable regions of gp120 are suitable for this alternative approach: (a) Cross-clade neutralizing Abs to the gp120 third variable region (V3) exist in patients[unreadable] sera;(b) Many human anti-V3 monoclonal Abs (mAbs) neutralize most Tier 1 and several Tier 2 viruses from various HIV-1 subtypes;and (c) Data from the Rv144 clinical vaccine trial suggest that Abs to another variable region, V2, may play a role in protection;and (d) human anti-V2 mAbs also display broad reactivity with gp120s from diverse clades and can mediate cross-clade neutralization. These data provide the rationale for developing immunogens that focus the immune response on V3 and V2 as a means of inducing Abs which, individually, may only display anti-viral activity against 20-40% of viruses, but together may inhibit a majority of diverse strains. Recently we have developed a platform for producing rationally-designed, recombinant V3-scaffold immunogens, and have used these as boosts after priming with gp120 DNA in rabbits to successfully elicit Ab responses that neutralize 10/11 Tier 1 viruses and 6/14 Tier 2 viruses from several clades. We now propose in Aim 1 to determine the optimal combination of envelope-based DNA primes and adjuvants for use with V3- scaffold immunogens, along with the best immunization schedule to induce broad, potent and long-lasting functional V3-specific Ab responses in rabbits. In Aim 2, we will use the platform developed with V3-scaffold immunogens to determine if V2-scaffold immunogens will induce broad, potent and long-lasting functional V2- specific Abs. For this, various V2- and V1V2-scaffold immunogens will be synthesized, tested for antigenicity in vitro, and for immunogenicity in vivo in rabbits. The optimized protocol developed in Aim 1 will be used for the in vivo experiments testing the V2-scaffold immunogens. In Aim 3, we will use the immunization regimen optimized in Aim 1 and the V3- and V2-scaffold immunogens developed and tested in Aims 1 and 2, to determine if induction of both V3 and V2 Abs results in additive or synergistic anti-viral activities in terms of potency and breadth. Thus, our overall goal is to develop and optimize an immunization regimen that induces long-lasting functional Abs with broad and potent anti-viral activity against HIV-1.